Pathologic or aberrant angiogenesis/neovascularization, aberrant remodeling, fibrosis and scarring and inflammation occur in association with retinal and ocular ischemic diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR) and in retinopathy of prematurity (ROP) and other developmental disorders (Eichler et al., 2006, Curr Pharm Des, 12: 2645-60) as well as being a result of infections and mechanical or chemical injury to the cornea and the eye in general (Ciulla et al., 2001, Curr Opin Opthalmol, 12: 442-9; Dart et al., 2003, Eye, 17: 886-92).
Diabetic retinopathy is a leading cause of blindness in adults of working age. The leading cause of vision loss for Americans under the age of 65 is diabetes; 16 million individuals in the United States are diabetic and 40,000 per year suffer from ocular complications of the disease, often a result of retinal neovascularization. DR, therefore, is a retinal microvascular disease that is manifested as a cascade of stages with increasing levels of severity and a worsening prognosis for vision. DR is broadly classified into 2 major clinical stages: nonproliferative diabetic retinopathy and proliferative diabetic retinopathy (PDR), where the term “proliferative” refers to the presence of preretinal neovascularization (PNV) emanating from the retina into the vitreous. Ocular neovascularization occurs in areas where capillary occlusions have developed, creating areas of ischemic retina and acting as a stimulus for neovascular proliferation that originate from pre-existing retinal venules at the optic disk and/or elsewhere in the retina posterior to the equator of the eye. Vitreous hemorrhage and tractional retinal detachment from PDR can cause severe vision loss (Boulton et al., 1997, Br J Opthalmol, 81: 228-223). Diabetic macular edema (DME) is another common cause of blindness (Levin, 2001, J Glaucoma 10:19-21; Stefansson et al., 1992, Am J. Opthalmol. 113:36-38). Clinical hallmarks of PDR include increased vascular permeability, leading to DME, and endothelial cell proliferation.
Age-related macular degeneration is a leading cause of vision loss in people over 65 years old. For example, AMD affects 12-15 million Americans over the age of 65 and causes vision loss in 10-15% of them. In contrast to ROP and PDR, in which neovascularization emanates from the retinal vasculature and extends into the vitreous cavity, AMD is associated with neovascularization originating from the choroidal vasculature and extending into the subretinal space. Choroidal neovascularization (CNV) causes severe vision loss in AMD patients because it occurs in the macula, the area of the retina responsible for central vision (Kitaoka et al., 1997, Curr Eye Res, 16:396-399).
Multiple theories exist, but the exact etiology and pathogenesis of AMD are still not well understood. Aging is associated with cumulative oxidative injury, thickening of Bruch's membrane and drusen formation. Oxidative stress results in injury to retinal pigment epithelial cells (RPE) and, in some cases, the choriocapillaris (Zarbin, 2004, Arch Opthalmol, 122: 598-614; Gorin et al., 1999, Mol V is, 5: 29). Injury to RPE likely elicits a chronic inflammatory response within Bruchs membrane and the choroid (Johnson et al., 2000, Exp Eye Res, 70: 441-9). This injury and inflammation fosters can potentiates retinal damage by stimulating CNV and atrophy (Zarbin, 2004, Arch Opthalmol, 122: 598-614; Witmer et al., 2003, Prog Retin Eye Res, 22: 1-29). CNV results in defective and leaky blood vessels (BV) that are likely to be recognized as a wound (Kent and Sheridan, 2003, Mol V is, 9: 747-55).
Retinopathy of prematurity (ROP) occurs most prominently in premature neonates. In various cases, the retina becomes completely vascularized at full term/near birth. In the premature baby, the retina remains incompletely vascularized at the time of birth. Rather than continuing in a normal fashion, vasculogenesis in the premature neonatal retina becomes disrupted. Maintaining the infants in incubators with high oxygen levels arrests the normal retinal vascular development and when they are removed to room air, this is a relative hypoxic environment and pathological angiogenesis results to compensate for the retinal oxygen deficiency due to insufficient vascularization. Abnormal new proliferating vessels develop at the juncture of vascularized and avascular retina. These abnormal new vessels grow from the retina into the vitreous, resulting in haemorrhage and tractional detachment of the retina (Neely et al., 1998, Am. J. Pathol, 153:665-670). It is estimated that visual impairment from this disease affects 3400 infants and causes blindness in 650 infants annually in the United States. Angiogenesis is the hallmark of this debilitating condition.
Others retinal diseases associated with retinal neovascularization include sickle cell retinopathy, retinal vein occlusion, certain inflammatory diseases of the eye, ocular tumorigenesis, Eale's disease, myopic choriodal neovascularization, and polypoidal choriodal vasculopathy. These, however, account for a much smaller proportion of visual loss caused by ocular neovascularization (Neely et al., 1998, American J. of Path. 153:665-670).
Corneal neovascularization, the abnormal formation of blood vessels in the cornea, is a common and serious complication of many corneal diseases and is a major cause of blindness that affects millions of people (Adamis, 2005, Retina, 25: 111-118). The condition is associated with severe visual impairment and is a high risk factor for graft rejection after allograft corneal transplantation. In addition, corneal neovascularization and subsequent opacification remain the most frequent causes of blindness after severe alkali burn trauma. To date, there are no pharmacological or surgical treatment options for the inhibition of corneal neovascularization that have been proven to be both safe and effective. Despite the routine use of topical steroids, the inflammatory response can lead to oedema, lipid deposition and corneal scarring that may not only significantly alter visual acuity, but also worsen the prognosis of subsequent penetrating keratoplasty. In addition, longer-term use of these drugs can lead to various adverse side effects such as cataracts, glaucoma, infection, and delayed corneal epithelial healing.
Angiogenesis is the process by which new blood vessels form (Carmeliet, 2005, Nature, 438: 932-936). In response to specific chemical signals, capillaries sprout from existing vessels, eventually growing in size as needed by the organism. Initially, endothelial cells, which line the blood vessels, divide in a direction orthogonal to the existing vessel, forming a solid sprout. Adjacent endothelial cells then form large vacuoles and the cells rearrange so that the vacuoles orient themselves end to end and eventually merge to form the lumen of a new capillary (tube formation).
Angiogenesis is stimulated by a number of conditions, such as in response to a wound, and accompanies virtually all tissue growth in vertebrate organisms such as mammals (Folkman, 2006, Annu Rev Med, 57: 1-18). In the normal adult, angiogenesis is tightly regulated, and is limited to wound healing, pregnancy and uterine cycling. Angiogenesis is turned on by specific angiogenic molecules such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), angiogenin, transforming growth factor, tumor necrosis factor-alpha. (TNF-alpha) and platelet derived growth factor. On the other hand, angiogenesis can be suppressed by inhibitory molecules such as interferon-α, thrombospondin-1, pigment epithelium derived factor (PEDF), angiostatin, and endostatin. It is the balance of these naturally occurring stimulators and inhibitors that controls the normally quiescent capillary vasculature. When this balance is upset, as in certain disease states, capillary endothelial cells are induced to proliferate, migrate and ultimately differentiate.
Angiogenesis plays a central role in a variety of diseases, including cancer and ocular neovascularization. Sustained growth and metastasis of a variety of tumors has also been shown to be dependent on the growth of new host blood vessels into the tumor in response to tumor derived angiogenic factors. Proliferation of new blood vessels in response to a variety of stimuli occurs as the dominant finding in the majority of eye diseases. In these diseases, tissue damage can stimulate release of angiogenic factors resulting in capillary proliferation (Gariano R F and Gardner T W, 2005, Nature, 438: 960-966). VEGF plays a dominant role in iris neovascularization and neovascular retinopathies. While reports clearly show a correlation between intraocular VEGF levels and ischemic retinopathic ocular neovascularization, FGF also likely plays an essential role. Basic FGF is known to be present in the normal adult retina, even though detectable levels are not consistently correlated with neovascularization. This may be largely due to the fact that FGF binds very tightly to charged components of the extracellular matrix and may not be readily available in a freely diffusible form that would be detected by standard assays of intraocular fluids. Furthermore, overexpression of bFGF in the eye does not stimulate neovascularization because it is sequestered (Ozaki et al., 1998, Am J Pathol, 153: 757-765), but bFGF does contribute to choriodal neovascularization when there is tissue disruption from the disease process itself or attempts at treatment (Yamada et al., 2000, J Cell Physiol, 185: 135-142).
Angiogenesis may be arrested or inhibited by interfering with the chemical signals that stimulate the angiogenic process. For example, angiogenic endothelial cells produce proteases to digest the basal lamina that surround the blood vessels, thus clearing a path for the new capillary. Inhibition of these proteases, or their formation, can prevent new vessels from forming Likewise, the endothelial cells proliferate in response to chemical signals. Particularly important proliferation signals include the VEGF and the bFGF families of proteins. Interference with these proliferation signaling processes can also inhibit angiogenesis.
Viable and approved current treatments for diseases related to ocular neovascularization are limited. The approved treatments for AMD are photodynamic therapy with VISUDYNE® (QLT/Novartis) and intravitreal injection of Macugen® (pegaptanib) (Eyetech/Pfizer) or Lucentis® (ranibizumab) (Genentech). Laser photocoagulation alone or photodynamic therapy with VISUDYNE® are therapies that involve laser-induced occlusion of the affected vasculature, which can result in localized damage to the retina. Macugen® (Eyetech/Pfizer) is an anti-VEGF aptamer that binds to VEGF165 preventing ligand-receptor interaction and is labeled for intravitreal injections every 4 weeks. Lucentis® (Genentech) is a humanized anti-VEGF antibody fragment that also binds directly to all isoforms of human VEGF and is labeled for intravitreal injections every 6 weeks. A variety of other pharmacologic therapies are undergoing clinical evaluation for AMD, such as RETAANE® 15 mg (anecortave acetate suspension, Alcon Research, Ltd.), Envision (squalamine, Genera), the VEGF R1R2 Trap, (Regeneron), Cand5 (anti-VEGF siRNA, Acuity), Sirna-027 (anti-VEGFR1 siRNA, SIRNA/Allergan), a topical receptor tyrosine kinase antagonist (TargeGen), sirolimus (rapamycin, MacuSight), etc.
Grid and pan retinal laser photocoagulation are the only proven options currently available for patients with diabetic macular edema or PDR, respectively. Multifocal laser photocoagulation may reduce retinal ischemia and inhibit angiogenesis by destroying healthy tissue and thus decreasing the total metabolic demand of the retina. Laser photocoagulation may also modulate the expression and production of various cytokines and trophic factors. Unfortunately, laser photocoagulation is a cytodestructive procedure and the visual field of the treated eye is irreversibly compromised. Surgical interventions, such as vitrectomy and removal of preretinal membranes, are widely used with or without laser treatment. Similar to the AMD trials, various pharmacologic agents are in clinical trials for DME, such as ARXXANT™ (ruboxystaurin mesylate, Lilly), RETISERT™ (fluocinolone acetonide, Bausch & Lomb), Posurdex (fluocinolone acetonide erodible implant, Occulex/Allergan), I-vation (nonerodible Dexamethasone implant, Occulex), Medidur (fluocinolone acetonide erodible implant, Alimera), etc. Intravitreal or periocular injection of triamcinolone acetonide, a corticosteroid (Kenalog®, Schering-Plough), and intravitreal Avastin® (anti-VEGF Mab (bevacizumab), Genentech) are also being used “off-label” for the treatment of both macular edema and wet AMD.
Anti-VEGF therapies represent a recent, significant advance in the treatment of exudative AMD. However, the phase III VISION Trial with PEGAPTANIB, a high affinity aptamer which selectively inhibits the 165 isoform of VEGF-A, demonstrated that the average patient continues to lose vision and only a small percent gained vision (Gragoudas et al., 2004, N Engl J Med, 351: 2805-16) Inhibition of all isoforms of VEGF-A (pan-VEGF inhibition) with the antibody fragment RANIBIZUMAB yielded much more impressive results (Brown et al., 2006, N Eng J Med, 355:1432-44; Rosenfeld et al., 2006, N Eng J Med 355:1419-31). The 2 year MARINA trial and the 1 year ANCHOR trial demonstrated that approximately 40% of patients achieve some visual gain. Although these results represent a major advance in our ability to treat exudative AMD, they also demonstrate that 60% of patients do not have visual improvement. Furthermore, these patients had to meet strictly defined inclusion and exclusion criteria. The results in a larger patient population may be less robust. In addition, adverse effects on neurons and vessels have been observed in primates after a single administration of the humanized anti-VEGF antibody (Bevacizumab) (Peters et al., 2007, Am J Opthalmol 91:827-31) and sporadic case reports of complications of anti-VEGF therapy related to regression of blood vessels, increased risk for stroke and myocardial infarction, and local side effects due to the intravitreal application mode have appeared (Fraunfelder et al., 2005, Drugs Today 41:703-9, Tobin et al., 2006, Insight 31:11-4, Rosenfeld et al., 2006, Opthalmol Clin NA 19:361-72, Baffert et al., 2006, Am J Physiol Heart Circ Physiol 290:H547-59, Hurwitz et al., 2004, Clin Colorectal Cancer 4 Suppl 2:S62-8). The limited efficacy and potential adverse effects of currently implemented therapies emphasize the need for alternative therapeutic strategies.
Ischemia-reperfusion injury (I/R injury) refers to an event in which the blood supply to a tissue is obstructed, such as myocardial infarction. Whenever there is a transient decrease or interruption of blood flow, the net injury is the sum of two components: the “direct” injury occurring during the ischemic interval and the “indirect” or reperfusion injury which follows. Reperfusion injury can be defined as the damage that occurs to an organ that is caused by the resumption of blood flow after an episode of ischemia. This damage is distinct from the injury resulting from the ischemia per se. One hallmark of reperfusion injury is that it may be attenuated by interventions initiated before or during the reperfusion. Reperfusion injury results from several complex and interdependent mechanisms that involve the production of reactive oxygen species, endothelial cell dysfunction, microvascular injury, alterations in intracellular Ca2+ handling, changes in tissue metabolism, and activation of neutrophils, platelets, cytokines and the complement system. All of the deleterious consequences associated with reperfusion constitute a spectrum of reperfusion-associated pathologies that are collectively called reperfusion injury. Reperfusion injury can extend not only acutely, but also over several days following the tissue attack.
During blood vessel obstruction, the endothelial tissue lining the affected blood vessels becomes “sticky” and begins to attract circulating white blood cells (Tohoku, 2008, J Exp Med, 215: 257-266). The white cells bound to the endothelium eventually migrate into the cardiac tissue causing significant tissue destruction. Although acute myocardial infarction is not directly caused by inflammation, much of the underlying pathology and the damage that occurs after an acute ischemia-reperfusion injury is caused by acute inflammatory responses during reperfusion, the restoration of blood flow to the affected myocardium. White blood cells present to the area, by the newly returning blood release, a host of inflammatory factors such as interleukins as well as free radicals in response to tissue damage. The restored blood flow reintroduces oxygen within cells that damages cellular proteins, DNA and the plasma membranes. Damage of the cell membrane may in turn causes release of more free radicals signaling apoptosis. Leukocytes may also build up in small capillaries, obstructing them and leading to more ischemia.
Cardiovascular disease is the leading cause of death in the western world. Coronary artery disease can lead to prolonged or irreversible episodes of cardiac ischemia that result in myocardial infarction (MI) which is associated with a high rate of mortality. The reduced blood flow in heart diseases is typically caused by blockage of a vessel by an embolus (blood clot); the blockage of a vessel due to atherosclerosis; the breakage of a blood vessel (a bleeding stroke); the blockage of a blood vessel due to vasoconstriction such as occurs during vasospasms and possibly, during transient ischemic attacks (TIA) and following subarachnoid haemorrhage. Conditions in which ischemia occurs further include myocardial infarction; trauma; and during cardiac and thoracic surgery and neurosurgery (blood flow needs to be reduced or stopped to achieve the aims of surgery). Procedures that can cause myocardial ischemia include coronary thrombolysis, coronary angioplasty (with or without stent placement), and coronary artery bypass grafts. During myocardial infarct, stoppage of the heart or damage occurs which reduces the flow of blood to myocardium, and ischemia results. Cardiac tissue itself is also subjected to ischemic damage. During various surgeries, reduction of blood flow, clots or air bubbles generated can lead to significant ischemic damage of the myocardium.
During an ischemic event, there is a gradation of injury that arises from the ischemic site. Cells at the site of blood flow restriction, undergo necrosis and form the core of a lesion. A penumbra is formed around the core where the injury is not immediately fatal, but progresses slowly toward cell death. This progression to cell death may be reversed upon reestablishment of blood flow within a short time of the ischemic event. Timely reperfusion to reduce the duration of ischemia is the definitive treatment to prevent cellular injury and necrosis in an ischemic myocardium. Typically reperfusion, after a short episode of myocardial ischemia (up to 15 min), is followed by the rapid restoration of cellular metabolism and function. Even with the successful treatment of occluded vessels, a significant risk of additional tissue injury after reperfusion may still occur. If the ischemic episode has been of sufficient severity and/or duration to cause significant changes in the metabolism and the structural integrity of heart muscle, reperfusion may paradoxically result in a worsening of heart function, out of proportion to the amount of dysfunction expected simply as a result of the duration of blocked flow. Although the beneficial effects of early reperfusion of ischemic myocardium with thrombolytic therapy, PTCA, or CABG are now well established, an increasing body of evidence indicates that reperfusion also induces an additional injury to ischemic heart muscle, such as the extension of myocardial necrosis, i.e., extended infarct size and impaired contractile function and metabolism. Hearts undergoing reperfusion after transplantation also undergo similar reperfusion injury events.
Despite efforts towards the development of new therapies for the treatment of diseases and conditions such as heart failure and cardiac ischemia/reperfusion injury, this remains an unmet need for additional or alternative agents to treat or prevent the onset or severity of this condition (Ferdinandy et al., 2007, Pharmacol Rev, 59: 418-458). Current therapies include the use of vasodilators, anti-thrombotics/thrombolytics, beta-blockers and coronary artery bypass graft, which are used pre and post myocardial ischemia to maintain/restore coronary blood flow and limit oxygen demand.